Laser scanning confocal microscopes have been known for many years, and provide the ability to image a relatively narrow focal plane within a sample. By then adjusting the position of the microscope with respect to the sample so as to take a slightly different focal plane, a “stack” of optically sectioned images can be obtained at different positions through the sample, thus allowing a three-dimensional image of the sample to be built up. An example laser scanning confocal microscope is shown in FIG. 5.
Here, a light source in the form of a laser beam 12 passes a light source aperture 14 and then is focused by an objective lens 18 into a small (ideally diffraction-limited) focal volume on a focal plane 20 within a fluorescent specimen 22. A mixture of emitted fluorescent light 24 as well as reflected laser light from the illuminated spot is then recollected by the objective lens 18. A dichroitic beam splitter 16 separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light 24 into the detection apparatus. After passing a pinhole 26 the fluorescent light is detected by a photo-detection device 28 (photomultiplier tube (PMT) or avalanche photodiode) transforming the light signal into an electrical one which is recorded by a computer (not shown).
The detector aperture obstructs the light that is not coming from the focal point, as shown by the light gray dotted line 30 in FIG. 5. The out-of-focus points are thus doubly suppressed: firstly they are less illuminated, and secondly most of their returning light is blocked by the pinhole. This results in sharper images compared to conventional fluorescence microscopy techniques and permits one to obtain images of various z axis planes (z-stacks) of the sample.
The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest a whole image is obtained pixel by pixel and line by line, while the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The beam is scanned across the sample in the horizontal plane using one or more (servo-controlled) oscillating mirrors. In comparison to the alternative, which is sample scanning, this scanning method usually has a low reaction latency. The scan speed can be varied. Slower scans provide a better signal to noise ratio resulting in better contrast and higher resolution. As mentioned, information can be collected from different focal planes e.g. by raising or lowering the microscope stage. The computer can then generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.
In order to obtain clearer and more detailed images it is desirable to try and increase the resolution of a confocal microscope. State of the art confocal systems achieve an increase in lateral (X-Y) resolution when the pinhole 26 is almost completely closed (e.g. <0.3 Airy units (AU)). However, such a technique leads to a severe loss in detected light intensity, whereas in microscopy and especially in fluorescence microscopy, the amount of detected light is precious.
In order to increase the amount of light available for imaging, therefore, within a typical application of a confocal laser scanning microscope for fluorescence detection the pinhole is opened to a diameter of >1 AU. Unfortunately, this then leads to a loss of resolution improvement along the in-plane (X-Y) directions, making the resolution of a confocal microscope with such a wide aperture of the pinhole almost identical to the X-Y resolution of a standard widefield microscope.
To overcome these conflicting problems it would be desirable to provide a technique which allows for the use of relatively wide pinholes (>0.3 AU), whilst preventing the attendant deleterious effects on the microscope resolution.